Methods and compositions for determining the purity of chemically synthesized nucleic acids

ABSTRACT

This application describes an antibody that specifically binds to a synthetic oligomer (e.g., an oligonucleotide or oligopeptide) having a organic protecting group covalently bound thereto, which antibody does not bind to that synthetic oligomer when the organic protecting group is not covalently bound thereto. Methods of making and using such antibodies are also disclosed, along with cells for making such antibodies and articles carrying immobilized oligomers that can be used in assay procedures with such antibodies.

RELATED APPLICATIONS

This application is a continuation-in-part of commonly owned, copendingapplication Ser. No. 09/476,975, filed Dec. 31, 1999, the disclosure ofwhich is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention concerns the detection, identification andquantification of protecting groups remaining after chemical synthesisof oligomers, particularly oligonucleotides.

BACKGROUND OF THE INVENTION

Over the past decade automated chemical synthesis of nucleic acids suchas DNA and RNA on solid supports has been developed. These chemicalprocesses include the use of agents to protect the exocyclic amines ofthe nucleotide bases adenine, thymine, cytosine and guanine and todirect the synthesis by blocking the 2′OH of RNA's ribose. The baseswithin the nucleic acid product of the synthesis are deprotected uponcleavage of the nucleic acid from the solid support. However, the extentof base deprotection is not easily determined.

For example, after base deprotection of synthetic RNA, products stillcontain the 2′-dimethylsilyl tert-butyl group as a protection of the2′OH of the ribose moiety. This protecting group is removed carefully bychemical means so as not to effect the chemistry and structure of theRNA. However, the extent of deprotection of the 2′OH is not readilydetermined. The nucleic acid is purified by high pressure liquidchromatography or by gel electrophoresis. However, some of the unwantedproducts of the synthesis are complete nucleic acid sequences that stillcontain one or more protecting groups, and shorter than full length(aborted) sequences difficult to separate from full length sequences,especially for oligomers of longer than 50 nucleosides. At present,there is no easy method to determine how much of each protecting group,if any, still remains on the product, and what proportion of the productis full-length. See generally Davis, G. E., Gehrke, C. W., Kuo, K. C.,and Agris, P. F. (1979) Major and Modified Nucleosides in tRNAHydrolysates by High Performance Liquid Chromatography. J. Chromatogr.173:281-298; Agris, P. F., Tompson, J. G., Gehrke, C. W., Kuo, K. C.,and Rice, R. H. (1980) High-Performance Liquid Chromatography and MassSpectrometry of Transfer RNA Bases for Isotopic Abundance. J.Chromatogr. 194:205-212; Gehrke, C. W., Kuo, K. C., McCune, R. A.,Gerhardt, K. O., and Agris, P. F. (1981) Quantitative EnzymaticHydrolysis of tRNAs: RP-HPLC of tRNA Nucleosides. J. Chromatogr.230:297-308; Chromatography and Modification of Nucleosides Volumes A, Band C (Gehrke, C. W. and Kuo, K. C. T., eds.), Elsevier Publishing Co.1990; Agris, P. F. and Sierzputowska-Gracz, H. (1990) Three DimensionalDynamic Structure of tRNA's by Nuclear Magnetic Resonance. InChromato-graphy and Modification of Nucleosides (Gehrke, C. W. and Kuo,K. C. T., eds.), Elsevier Publishing Co., pp. 225-253; Agris, P. F.,Hayden, J., Sierzputowska-Gracz, H., Ditson, S., Degres, J. A.,Tempesta, M., Kuo, K. C. and Gehrke, C. W. (1990) Compendium onBiological, Biochemical, Chemical, Physical and Spectroscopic Propertiesof RNA and DNA Nucleosides. In Chromatography and Modification ofNucleosides, Elsevier Publishing Co.

The incomplete removal of the protecting group and lack of a simpleassay is a problem for two industries and for numerous researchers worldwide: (i) the multitude of companies now providing nucleic acid sequencesynthesis products by overnight delivery have difficulty telling theircustomers the extent to which the product is deprotected; (ii)pharmaceutical companies cannot easily verify for regulatory agenciesthe purity and/or length of the therapeutic or diagnosticoligonucleotide products they seek to introduce or market. Accordingly,there is a need for simple and reliable techniques for determining thepurity and proportion of full length of oligonucleotide products.

SUMMARY OF THE INVENTION

A first aspect of the present invention is an antibody (e.g., amonoclonal or polyclonal antibody) that specifically binds to asynthetic oligomer (i.e., an oligonucleotide or oligopeptide) having aorganic protecting group covalently bound thereto, which antibody doesnot bind to that synthetic oligomer when the organic protecting group isnot covalently bound thereto.

A second aspect of the present invention comprises a cell or cells,including cell cultures and isolated cells, that express an antibody asdescribed above. Such cells include hybridoma cells, as well asrecombinant cells that contain and express a heterologous nucleic acidencoding the antibody.

A third aspect of the present invention is a method for detectingincomplete deprotection of a synthetic oligomer by immunoassay, saidimmunoassay comprising the steps of: (a) contacting a synthetic oligomerto an antibody as described above, and then (b) detecting the presenceor absence of binding of said antibody to said oligomer, the presence ofbinding indicating incomplete deprotection of said synthetic oligomer.Any suitable assay format can be employed, including heterogeneous andhomogeneous immunoassays. For example, the immunoassay may be animmunoblot-dot assay, or may be a sandwich assay.

A fourth aspect of the present invention is a method for separatingprotected (including partially and completely protected) syntheticoligomers from fully deprotected synthetic oligomers. The methodcomprises (a) contacting a mixture of protected from fully deprotectedsynthetic oligomers to antibodies as described above, wherein theprotected synthetic oligomers have the organic protecting groupcovalently bound thereto, so that the protected synthetic oligomers bindto the antibody; and then separating the antibodies from the fullydeprotected oligomers. The antibody may be immobilized on a solidsupport to facilitate separation. The protected synthetic oligomer maybe a partially protected synthetic oligomer (for which one applicationis the identification and/or purification of full-length versus abortedsequence oligomers) or a fully protected synthetic oligomer that has notundergone deprotection. Any separation format may be used, including butnot limited to affinity chromatography.

A fifth aspect of the invention is an article useful for the determiningincomplete deprotection of a synthetic oligomer in an immunoassay, saidarticle comprising: (a) a solid support (e.g., a nitrocellulose strip)having a surface portion, said surface portion having at least twoseparate discrete regions formed thereon; (b) a first oligomer bound toone of said separate discrete regions, said first oligomer having aprotecting group bound thereto; and (c) a second oligomer bound toanother of said separate discrete regions, said second oligomer nothaving said protecting group bound thereto; wherein the nucleotidesequence of said first and second oligomers are the same. In a preferredembodiment, the article further comprises (d) a third oligomer bound toanother of said separate discrete regions; said third oligomer alsohaving said protecting group bound to said first oligomer bound thereto;wherein said third oligomer is partially deprotected; and wherein thenucleotide sequence of said first, second, and third oligomers are thesame.

A sixth aspect of the present invention is a method of making anantibody that specifically binds to a synthetic oligomer having aorganic protecting group covalently bound thereto, which antibody doesnot bind to the said synthetic oligomer when said organic protectinggroup is not covalently bound thereto, said method comprising the stepsof: (a) synthesizing said synthetic oligomer on a solid particulatesupport (and preferably covalently bound thereto, e.g., with a succinyllinker) with said organic protecting group covalently bound to saidsynthetic oligomer (or synthesizing a monomer of a single nucleotide onthe solid support, with the single nucleotide having said protectinggroup covalently bound thereto); and then, without removing saidoligomer from said solid support; (b) immunizing an animal with saidsynthetic oligomer bound to said solid support (or monomer bound to saidsolid support) in an amount sufficient to produce said antibody.Optionally, the solid support can be replaced with a carrier group suchas a protein (e.g., bovine serum albumin).

In summary, the antibodies and methods of the present invention areuseful in immunoassays, such as for the qualitative and quantitativedetection of protecting groups used in organic synthetic processes, withparticular application to oligonucleotides or peptides in research,therapeutics, diagnostics and biomedical science. The antibodies of theinvention can be used in purification techniques, such as for theseparation of final products from by-product contaminants. The instantinvention can be used in the course of quality control ofoligonucleotide and peptide synthesis, such as in the quality control ofdrugs for gene therapy, antisense, antigene and control of geneexpression, in the quality control of biomedical polymers that maycontain protecting groups, and as probes for purification andcharacterization of synthetic oligomers, particularly oligonucleotidesor peptides.

The present invention is explained in greater detail in the drawingsherein and the specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a dot-blot immunoassay of monoclonal antibody 1 H11, whichselectively binds to oligoIbu-dG20mers.

FIG. 2 is a dot-blot immunoassay of monoclonal antibody 7H3, whichselectively binds to oligoBz-dC20mers.

FIG. 3 shows ELISA (A) and dot-blot (B) results demonstratingspecificity and detection sensitivity of a monoclonal antibody (mAb) ofthe commonly used protecting group, benzoyl (Bz), for the chemicalsynthesis of nucleic acids. Partially deprotected oligomer oligo Bz-dC(center column) can be re-treated to remove the remaining protectinggroups, and re-tested with mAb (C). An RNA standard with protectinggroups Bz, ibu and ipr-Pac was synthesized and assayed foridentification of the protecting groups with the mAb against Bz (D).

FIG. 4 shows ELISA (A) and dot-blot (B) results demonstratingspecificity and sensitivity of a monoclonal antibody (mAb) and itsdetection of the commonly used protecting group, isobutryl (ibu), forthe chemical synthesis of nucleic acids. Dot-blot assay with highamounts of DNA demonstrates that the ibu protecting group was recognizedby the mAb no matter which nucleobase was protected (C). Partiallydeprotected oligomer oligo Bz-dC (center column) can be re-treated toremove the remaining protecting groups, and re-tested with mAb (D). AnRNA standard with protecting groups Bz, ibu and ipr-Pac was synthesizedand assayed for identification of the protecting groups with the mAbagainst ibu (E).

FIG. 5 shows ELISA (A) and dot-blot (B) results demonstratingspecificity and sensitivity of a monoclonal antibody (mAb) and itsdetection of the commonly used protecting group, isopropylphenoxyacetyl(ipr-Pac), for the chemical synthesis of nucleic acids. Partiallydeprotected oligomers oligo ibr-Pac-dG and oligo ibu-dG (columns secondfrom left and forth from left, respectively) can be re-treated to removethe remaining protecting groups, and re-tested with mAb (C). An RNAstandard with protecting groups Bz, ibu and ipr-Pac was synthesized andassayed for identification of the protecting groups with the mAb againstipr-Pac (D).

FIG. 6 shows a mAb dot-blot assay of protecting groups demonstrating thesensitivity and quantifiable response of the technology as related toHPLC. Dot-blot detection of Bz groups remaining on a standardized 20meroligo dC molecule was analzyed (A) and a quantitation of the mAbresponse (B) was determined. The mAb response was analyzed with anincrease in the amount of DNA on the dot-blot membrane (C). The columnon the left is just the protected Bz-dC 20mer. The column on the rightis the protected Bz-dC together with a 2500-fold excess of thecompletely deprotected oligo dC(Bz).

FIG. 7 shows a direct comparison of the mAb and HPLC detection of Bz inthe pmole (A) and nmol range (B), respectively.

FIG. 8 shows a blind study demonstrating the detection of remainingprotecting groups in commercial samples. dA-dC oligos were analyzed withanti-Bz mAb (A) and dG-dT oligos were analyzed with anti-ipr-Pac mAb(B). The oligo dA-dC samples from companies #2 and #6 were tested inhigher amounts to confirm the presence of the Bz protecting group (C).In addition, the samples were treated to remove the remaining protectinggroups using a standard protocol. The oligo dG-dT samples were assayedfor the ipr-Pac protecting groups (D). The samples were re-treated toremove remaining protecting groups and re-analyzed as in (C).

FIG. 9 shows the production and analyses of polycolonal antibody againstthe 5′ terminal protecting group, dimethyltrityl (DMT).

FIG. 10 shows a substrate carrying different oligonucleotides of thesame sequence, but with varying degrees of deprotection, that may beused as a testing standard to screen similar oligonucleotides of thesame sequence for varying degrees of protection or deprotection.

FIG. 11 illustrates an oligonucleotide array that may be screened forthe presence of protecting groups or insufficient elongation withantibodies of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 1. General Definitions

“Antibody” as used herein refers to both monoclonal and polyclonalantibodies, refers to antibodies of any immunoglobulin type (includingbut not limited to IgG and IgM antibodies), and including antibodyfragments that retain the hypervariable or binding regions thereof.Antibodies may be of any species of origin, but are typically mammalian(e.g., horse, rat, mouse, rabbit, goat). Antibodies may be bound to orimmobilized on solid supports such as nitrocellulose, agarose, glass,organic polymers (“plastics”) and the like in accordance with knowntechniques, and may be labeled with or joined to other detectable groupsin accordance with known techniques.

“Binding” as used herein with respect to the selective binding of anantibody to an oligomer has its usual meaning in the art. In general, toobtain useful discrimination in an immunoassay or an affinitypurification technique, the antibody should bind to the protectedoligomer at an affinity of at least about k_(d)=10⁻⁶, 10⁻⁷, or 10⁻⁸ M,and should bind to the unprotected oligomer at an affinity of notgreater than about k_(d)=10⁻², 10⁻³, or 10⁻⁴ M.

“Oligomer” as used herein refers to synthetic oligonucleotides andsynthetic oligopeptides, including synthetic oligomers in the naturallyoccurring form such as DNA and RNA, and modified backbone chemistries asdiscussed below. Oligonucleotides are currently preferred in carryingout the present invention, and the instant invention is primarilyexplained with reference to oligonucleotides herein. However, themethods and techniques described herein may also be applied tooligopeptides, oligosaccharides, etc. (i.e., any synthetically producedpolymer requiring protecting groups for synthesis).

“Nucleotide” as used herein refers to a subunit of an oligonucleotidecomprising a pentose, a nitrogenous heterocyclic base (typically boundto the 1 position of the pentose), and a phosphate or phosphoric acidgroup (typically bound at the 5′ position of the pentose) but absent, orconsidered bound at the 3′ position, in the 5′ terminal nucleotide of anoligonucleotide. These structures are well known. See, e.g., A.Lehninger, Biochemistry, 309-320). “Nucleoside” typically refers to anucleotide, absent a phosphoric acid or phosphate group.

“Protecting group” as used herein has its conventional meaning in theart and refers to a chemical moiety, group or substituent that iscoupled, typically covalently coupled, to an atom in a molecule prior toa chemical reaction involving that molecule (typically in an organicsynthesis), so that the chemical reaction is averted at the atom towhich the protecting group is coupled. Typically, the protecting groupis then chemically removed from the intermediate molecule forpreparation of the final product, although removal techniques may not beentirely successful leading to only partial deprotection of the finalproduct (i.e., the presence of at least one protecting group remainingon that molecule). Protecting groups may be intentionally left on amolecule for purposes of generating or testing an antibody as describedherein.

“Deprotection” or “deprotected” as used herein refers to the absence ofprotecting groups employed during chemical oligonucleotide synthesisfrom a molecule. Such protecting groups are described below. Thepresence of such a protecting group may indicate insufficient elongationof the oligonucleotide, when the protecting group is chain terminating.Chemically synthesized oligonucleotides are ideally fully deprotected,but the present invention is employed to detect partial or incompletedeprotection of such oligonucleotides (that is, the presence of at leastone protecting group as described below in the oligonucleotide).

“Base” as used herein with respect to oligonucleotides refers to anitrogenous heterocyclic base which is a derivative of either purine(e.g., adenine, guanine) or pyrimidine (e.g., uracil, thymine,cytosine). Pyrimidine bases are bound to the pentose by the 1 ringnitrogen; Purine bases are bonded to the pentose by the 9 ring nitrogen.Preferred bases are those that contain a free amino group, such asguanine, adenine, and cytosine (the protecting group is then covalentlybound to the free amino group by substitution of one, or both, of thehydrogens on the free amino group). However, the present invention maybe used with any purine or pyrimidine base, whether standard ormodified/rare, that contains a free amino group for protection, or othergroup requiring protection during synthesis thereof in anoligonucleotide. Examples of standard and modified/rare bases are thosefound in the nucleosides set forth in Table 1 below.

TABLE 1 Standard and modified nucleosides and their standardabbreviations. abbreviation base U uridine C cytidine A adenosine Gguanosine T thymidine ?A unknown modified adenosine m1A1-methyladenosine m2A 2-methyladenosine i6A N⁶-isopentenyladenosinems2i6A 2-methylthio-N⁶-isopentenyladenosine m6A N⁶-methyladenosine t6AN⁶-threonylcarbamoyladenosine m6t6AN⁶-methyl-N⁶-threonylcarbomoyladenosine ms2t6A2-methylthio-N⁶-threonylcarbamoyladenosine Am 2′-O-methyladenosine IInosine m1I 1-methylinosine Ar(p) 2′-O-(5-phospho)ribosyladenosine io6AN⁶-(cis-hydroxyisopentenyl)adenosine ?C Unknown modified cytidine s2C2-thiocytidine Cm 2′-O-methylcytidine ac4C N⁴-acetylcytidine m5C5-methylcytidine m3C 3-methylcytidine k2C lysidine f5C 5-formylcytidinef5Cm 2′-O-methyl-5-formylcytidine ?G unknown modified guanosine Gr(p)2′-O-(5-phospho)ribosylguanosine m1G 1-methylguanosine m2GN²-methylguanosine Gm 2′-O-methylguanosine m22G N²N²-dimethylguanosinem22Gm N²,N²,2′-O-trimethylguanosine m7G 7-methylguanosine fa7d7Garchaeosine Q queuosine manQ mannosyl-queuosine galQgalactosyl-queuosine Yw wybutosine o2yW peroxywybutosine ?U unknownmodified uridine mnm5U 5-methylaminomethyluridine s2U 2-thiouridine Um2′-O-methyluridine s4U 4-thiouridine ncm5U 5-carbamoylmethyluridinemcm5U 5-methoxycarbonylmethyluridine mnm5s2U5-methylaminomethyl-2-thiouridine mcm5s2U5-methoxycarbonylmethyl-2-thiouridine cmo5U uridine 5-oxyacetic acidmo5U 5-methoxyuridine cmnm5U 5-carboxymethylaminomethyluridine cmnm5s2U5-carboxymethylaminomethyl-2-thiouridine acp3U3-(3-amino-3-carboxypropyl)uridine mchm5U5-(carboxyhydroxymethyl)uridinemethyl ester cmnm5Um5-carboxymethylaminomethyl-2′-O-methyluridine ncm5Um5-carbamoylmethyl-2′-O-methyluridine D Dihydrouridine ψ pseudouridinem1ψ 1-methylpseudouridine ψm 2′-O-methylpseudouridine m5U ribosylthyminem5s2U 5-methyl-2-thiouridine m5Um 5,2′-O-dimethyluridine

See Sprinzl et al., Nucleic Acids Res. 26, 148 (1998).

Applicants specifically intend that the disclosures of all United Statespatent references cited herein be incorporated by reference herein intheir entirety.

2. Protecting Groups

The particular protecting group will depend upon the oligomer beingsynthesized and the methodology by which that oligomer is synthesized.

For the synthesis of oligonucleotides, suitable protecting groupsinclude alkyl, aryl, alkylaryl, arylalkyl groups, which may contain oneor more hetero atoms such as N, O, or S, and which may be substituted orunsubstituted (e.g., a carbonyl group). Examples of protecting groupsinclude; but are not limited to, the following: acetyl; isobutyryl;2-(t-butyldiphenyl-silyloxymethyl)benzoyl; naphthaloyl;iso-butyryloxycarbonyl; levulinyl; fluorenylmethoxycarbonyl;2-nitrothiophenyl; 2,2,2-trichloro-t-butoxycarbonyl, ethoxycarbonyl;benzyloxycarbonyl; p-nitrophenyl-ethyloxycarbonyl;N′N-dimethylformamidine; formyl; benzoyl, toluyl;2,4-6-trimethylbenzoyl; anisoyl; 2,4-dimethylphenyl;2,4,6-trimethylphenyl; triphenylthiomethyl; pivoloiloxymethyl;t-butoxycarbonyl; p-nitrophenylethyl; methoxyethoxymethyl;butylthiocarbonyl; 2-methyl-pyridine-5-yl; 2-nitrothiophenyl;2,4-dinitrothiophenyl; 2-nitro-4-methylthiophenyl;p-nitrophenylsulphonylethyl; 5-chloro-8-hydroxyquinoline; thiophenyl;β-cyanoethyl; phenylethyl; p-nitrophenylethyl; pyridylethyl;2-N-methylimidazolylphenyl; methyl; allyl; trichloroethyl; dibenzoyl;p-nitrophenylethoxycarbonyl; benzoyl and substituted derivativesthereof; 2(acetoxymethyl) benzoyl; 4,4′,4″-tris-(benzyloxy)trityl;5-methylpyridyno-2-yl; phenylthioethyl; dipehylcarbamoyl;3,4-dimethoxybenzyl; 3-chlorophenyl; 2-nitrophenyl;9-pnenylxanthen-9-yl; 9-(p-methoxyphenyl)xanthen-9-yl;9-(p-ocatadecyloxyphenyl)xanthen-9-yl; “bridged” bis-dimethoxytritylgroups; phthaloyl; succinyl; benzensulphonylethoxycarbonyl;4,4′,4″-tris(bevulinyloxy)trityl; p-phenylazophenyloxycarbonyl;o-substituted benzoyl; 4,4′ 4″-tris-(4,5-dichlorophalimidin)trityl;levelinyl; alkyloxy and aryloxyacetyl; 1,3-benzodithiol-2-yl;tetrahydrofuranyl; [2-(methylthio)phenyl]thiomethyl;1-(2-chloroethyoxy)ethyl; 1-[(2-fluoro-phenyl]4-methoxy piperidin-4-yl;4-methoxytetrahydropyran-4-yl; (1-methyl-1-methoxy)ethyl;tetrahydropyranyl; 3-methoxy-1,5-dicarbomethoxypentam-3-yl;2-nitrobenzyl; benzyl; 4-nitrophenylethyl-sulphonyl;t-butyldimethylsilyl; 4-methoxybenzyl; 3,4-dimethoxybenzyl;9-p-methoxyphenylthioxanthen-9-yl; compounds of the formula R₁R₂R₃C—,wherein R₁, R₂, and R₃ are each independently selected from the groupconsisting of phenyl, p-monomethoxyphenyl, o-monomethoxphenyl, biphenyl,p-fluoropnehyl, p-chlorophenyl, p-methylphenyl, p-nitrophenyl, etc.

3. Oligonucleotides

Synthetic oligonucleotides that contain protecting groups and may beused to carry out the present invention include both the naturallyoccurring forms such as DNA and RNA, and those with modified backbonechemistries, such as poly (phosphate derivatives) such as phosphonates,phosphoramides, phosphonamides, phosphites, phosphinamides, etc., poly(sulfur derivatives) e.g., sulfones, sulfonates, sulfites, sulfonamides,sulfenamides, etc. It will be noted that antibodies of the invention maybe characterized by their selective binding to particular “reagent” or“benchmark” oligonucleotides, but the same antibodies may also bind to avariety of other oligonucleotides (e.g., longer nucleotides) or othercompounds that contain the same protecting group.

For example, an oligonucleotide to which the antibody selectively bindsmay consist of from 3 to 20 nucleotides, and wherein one of saidnucleotides is a protected nucleotide according to Formula (I) below:

wherein:

R is H or a protecting group, such as dimethoxytrityl; subject to theproviso that R is a covalent bond to an adjacent nucleotide when saidprotected base is not a 5′ terminal nucleotide in said oligonucleotide;

R₁ is H or a protecting group such as β-cyanoethyl; subject to theproviso that R₁ is a covalent bond to an adjacent nucleotide when saidprotected base is not a 3′ terminal nucleotide in said oligonucleotide;

R₂ is H or —OR₃;

R₃ is H or a protecting group such as tert-butyldimethylsilyl;

Base is a purine or pyrimidine base; and

R₄ is a protecting group bonded to an amino group of said base, such asa protecting group is selected from the group consisting of acetyl (Ac),benzoyl (Bz), dimethylformamidine (dmf), isobutyrl (Ibu), phenoxyacetyl(Pac), and isopropyl-phenoxyacetyl (Ipr-pac);

and further subject to the proviso that when one of R, R₁, R₃ and R₄ isa protecting group, then the others of R, R₁, R₃ and R₄ are notprotecting groups.

In one particular embodiment of the foregoing, the antibody may be onethat selectively binds to an oligonucleotide that consists of from 3 to20 nucleotides and has a 5′ nucleotide, and wherein said 5′ nucleotideis a protected nucleotide according to Formula (I):

wherein:

R is a protecting group such as dimethoxytrityl;

R₁ is a covalent bond to an adjacent nucleotide;

R₂ is —H or —OH; and

Base is a purine or pyrimidine base.

In another particular embodiment of the foregoing, the antibody may beone that selectively binds to an oligonucleotide that consists of from 3to 20 nucleotides and has a 3′ nucleotide, and wherein said 3′nucleotide is a protected nucleotide according to Formula (I):

wherein:

R is a covalent bond to an adjacent nucleotide;

R₁ is a protecting group such as β-cyanoethyl;

R₂ is H or —OH; and

Base is a purine or pyrimidine base.

In another particular embodiment of the foregoing, the antibody may beone that selectively binds to an oligonucleotide that consists of from 3to 20 nucleotides, and wherein one of said nucleotides is a protectednucleotide according to Formula (I):

wherein:

R is a covalent bond to an adjacent nucleotide;

R₁ is a covalent bond to an adjacent nucleotide;

R₂ is —OR₃;

R₃ a protecting group such as tert-butyldimethylsilyl; and

Base is a purine or pyrimidine base.

In still another particular embodiment of the foregoing, the antibodymay be one that selectively binds to an oligonucleotide that consists offrom 3 to 20 nucleotides, and wherein one of said nucleotides is aprotected nucleotide according to Formula (I):

wherein:

R is a covalent bond to an adjacent nucleotide;

R₁ is a covalent bond to an adjacent nucleotide;

R₂ H or —OH;

Base is a purine or pyrimidine base; and

R₄ is a protecting group bonded to an amino group of said base, such asacetyl, benzoyl, dimethylformamidine, isobutyryl, phenoxyacetyl, andisopropyl-phenoxyacetyl.

Thus, examples of protected bases that may be employed in the structuresshown above include, but are not limited to, adenine, guanine, andcytosine, as follows:

wherein R₁ and R₂ are both H in an unprotected base, and either R₁ or R₂are a protecting group as described above (e.g. Pac, Ipr-pac, Ibu, Bz,Ac, dmf) for a protected base. Likewise, modified nucleosides haveprotecting groups at the modifications that are chemically reactive.

In one embodiment of the invention, the oligonucleotides are peptidenucleic acids, and the protecting groups are those protecting groupsemployed in the synthesis of peptide nucleic acids, includinb but notlimited to those described in U.S. Pat. No. 6,133,444.

In still another particular embodiment of the foregoing, the antibodymay be one that selectively binds to an oligonucleotide that consists offrom 3 to 20 nucleotides, and wherein one of said nucleotides is aprotected with a photolabile protecting group, including but not limitedto those described in U.S. Pat. Nos. 5,744,101 and 5,489,678 (assignedto Affymax).

4. Antibodies

As noted above, the present invention provides antibodies (e.g., amonoclonal or polyclonal antibody) that specifically bind to a syntheticoligonucleotide having a organic protecting group covalently boundthereto, which antibody does not bind to said synthetic oligonucleotidewhen said organic protecting group is not covalently bound thereto.

The antibody may be provided immobilized on (or bound to) a solidsupport in accordance with known techniques, or may be provided in afree, unbound form (e.g., lyophilized, frozen, in an aqueous carrier,etc.). Whether or not an antibody is immobilized will depend upon theparticular immunoassay or affinity purification technique in which theantibody is used, and is determined by the known parameters for suchtechniques. Similarly, the antibody may be bound to or conjugated withsuitable detectable groups, such as an enzyme (e.g., horseradishperoxidase), a member of a binding pair such as biotin or avidin, aradioactive group or a fluorescent group such as green fluorescentprotein, also in accordance with known techniques, typically dependingupon the immunoassay format in which the antibody is used.

5. Immunoassay Methods

The present invention provides a method for detecting incompletedeprotection of a synthetic oligonucleotide (including aborted sequencesthat still contain a protecting group) by immunoassay. In general, suchan immunoassay comprises the steps of: (a) contacting a syntheticoligonucleotide to an antibody as described above, and then (b)detecting the presence or absence of binding of said antibody to saidoligonucleotide, the presence of binding indicating incompletedeprotection of said synthetic oligonucleotide. Any suitable assayformat can be employed, including heterogeneous and homogeneousimmunoassays. For example, the immunoassay may be an immunoblot-dotassay, or may be a sandwich assay. The oligonucleotides being tested fordeprotection may be in any suitable form, such as in solution orimmobilized on a solid support.

In a preferred embodiment, the detection method employs a “dip stick” orthe like, in which binding of the antibody to the test oligonucleotideis compared to binding of the antibody to a set of knownoligonucleotides, all immobilized on a common solid support. Such anarticle, as illustrated in FIG. 10, useful for determining incompletedeprotection of a synthetic oligonucleotide in an immunoassay,comprises: (a) a solid support (e.g., a nitrocellulose strip) 25 havinga surface portion, said surface portion having at least two separatediscrete regions 26, 27 formed thereon; (b) a first oligonucleotidebound to one of said separate discrete regions, said firstoligonucleotide having a protecting group bound thereto (e.g., at leastone protecting group); and (c) a second oligonucleotide bound to anotherof said separate discrete regions, said second oligonucleotide nothaving said protecting group bound thereto; wherein the nucleotidesequence of said first and second oligonucleotides are the same. In apreferred embodiment, the article further comprises (d) a thirdoligonucleotide bound to another of said separate discrete regions 28;said third oligonucleotide also having said protecting group bound tosaid first oligonucleotide bound thereto; wherein said thirdoligonucleotide is partially deprotected (i.e., has a number ofprotecting groups covalently bound thereto which is intermediate betweenthat bound to the first and second oligonucleotide, e.g., at least one,two three or four more protecting groups than the first oligonucleotide,up to at least 10, 20 or more protecting groups than the firstoligonucleotide); and wherein the nucleotide sequence of said first,second, and third oligonucleotides are the same. Of course, still moreoligonucleotides carrying varying numbers of protecting groups may beincluded on the substrate in additional separate and discrete locations,if desired. The discrete regions to which the separate oligonucleotidesare bound may be in any form, such as dots.

6. Affinity Purification Methods

In addition to immunoassays, the present invention also providesaffinity purification techniques for the separation of fully deprotectedoligonucleotides from partially deprotected (including fully protected)oligonucleotides (e.g., both oligonucleotides that have been subjectedto a deprotection process to remove the protecting group, andoligonucleotides that have not). Such a procedure typically comprises(a) contacting a mixture of protected and fully deprotected syntheticoligonucleotides to antibodies as described above, wherein the protectedsynthetic oligonucleotides have the organic protecting group for whichthe antibody is selective covalently bound thereto, so that theprotected synthetic oligonucleotides bind to the antibody; and thenseparating said antibodies from said fully deprotected oligonucleotides.The antibody may be immobilized on a solid support to facilitateseparation. The protected synthetic oligonucleotide may be a partiallyprotected synthetic oligonucleotide, or a fully protected syntheticoligonucleotide that has not undergone deprotection. Any separationformat may be used, including but not limited to affinitychromatography.

7. Production of Antibodies

A method of making an antibody that specifically binds to a syntheticoligonucleotide having a organic protecting group covalently boundthereto, which antibody does not bind to the said syntheticoligonucleotide when said organic protecting group is not covalentlybound thereto, comprises the steps of: (a) synthesizing the syntheticoligonucleotide on a solid particulate support (and preferablycovalently bound thereto, e.g., with a succinyl linker) with the organicprotecting group covalently bound to said synthetic oligonucleotide; andthen, without removing the oligonucleotide from said solid support; and(b) immunizing an animal with the synthetic oligonucleotide bound to thesolid support in an amount sufficient to produce the antibody. Inaddition, a single nucleotide can be bound to the solid particulatesupport with the organic protecting group bound thereto, and used asdescribed hereinabove.

The synthesis step may be carried out on the solid support in accordancewith known techniques. The solid support may be in particulate formprior to synthesis, or may be fragmented into particles after synthesis.In general, the solid supports are beads, which may be completely solidthroughout, porous, deformable or hard. The beads will generally be atleast 10, 20 or 50 to 250, 500, or 2000 μm in diameter, and are mosttypically 50 to 250 μm in diameter. Any convenient composition can beused for the solid support, including cellulose, pore-glass, silica gel,polystyrene beads such as polystyrene beads cross-linked withdivinylbenzene, grafted copolymer beads such aspolyethyleneglycol/polystyrene, polyacrylamide beads, latex beads,dimethylacrylamide beads, composites such as glass particles coated witha hydrophobic polymer such as cross-linked polystyrene or a fluorinatedethylene polymer to which is grafted linear polystyrene, and the like.Where separate discrete solid supports such as particles or beads areemployed, they generally comprise from about 1 to 99 percent by weightof the total reaction mixture.

In a preferred embodiment, the synthesizing step is followed by the stepof fragmenting the solid support (e.g., by crushing) prior to theimmunizing step. Polyclonal antibodies may be collected from the serumof the animal in accordance with known techniques, or spleen cells maybe collected from the animal, a plurality of hybridoma cell linesproduced from the spleen cells; and then a particular hybridoma cellline that produces the antibody isolated from the plurality of hybridomacell lines.

A particular protocol for the production of antiserum/polyclonalantibodies and monoclonal antibodies against protecting groups used innucleic acid and other synthesis typically involves the following steps:(a) preparation of oligonucleotides and others that contain or do notcontain protecting groups; (b) immunization of animals with thosepreparations; (c) screening of animals to identify those that exhibitantibodies against protecting groups; (d) production of monoclonalantibody by classical fusion method; (e) optionally, production ofscFab, Fab fragments and whole antibody molecules by antibodyengineering; and (f) evaluation and characterization of monoclonalantibodies against the protecting groups. Each of these steps isdiscussed in greater detail below.

Synthetic oligonucleotides that contain protecting groups can besynthesized in a variety of ways known to those skilled in the art. Forexample, protecting groups can be attached to individual nucleotidesthat are linked to controlled pore glass (CPG) beads. An example is:

CPG bead---dT (only with DMT group).

In the alternative, protecting groups may be attached to oligonucleotidechains that are linked to CPG beads. Examples include:

Pac-dA---Pac-dA---CPG beads with Bz-dC and Ibu-dG;

Ipr-Pac-dG---Ipr-Pac-dG---CPG beads with Bz-dC and Ibu-dG;

Ac-dC---Ac-dC---CPG beads with Bz-dC and Ibu-dG;

dmf-G---dmf-G---CPG beads with Bz-dC and Ibu-dG; and

mixtures of the four oligonucleotides described above.

In another alternative, protecting groups may be attached tooligonucleotide chains that are partially deprotected (the procedure fordeprotection will be described bellow). Examples include:

Poly dT20mers (only with DMT group);

Poly dT20mers (only with cyanoethyl groups);

Poly Ibu-dG 20mers (partially deprotected);

Poly Ipr-Pac-dG 20mers (partially deprotected);

Poly Bz-dC 20mers (partially deprotected);

Poly Pac-dA 20mers (partially deprotected); and

Poly Ac-dC 20mers (partially deprotected).

Synthetic oligonucleotides prepared as described herein may be partiallydeprotected as follows: (a) add 30% ammonium hydroxide solution tosynthetic polynucleotides, then incubate at room temperature fordifferent time periods (5, 10 and 30 min); (b) take the ammoniumsolution of treated oligomers and add into 1:1 diluted acetic acidpre-cooled at 4° C. and according to 1:4 ratio of ammonium to aceticacid; (c) keep samples in ice bath for 30 min; (d) dry samples withspeed-Vac; (e) dissolve the dried pellets in water; (f) desalt sampleswith Sephadex G-25 column; (g) dry samples with speed-Vac; and (h)dissolve the desalted samples in water.

Synthetic oligonucleotides prepared as described herein may becompletely deprotected by any suitable technique. One particulartechnique is as follows: (a) add 30% ammonium hydroxide solution tosynthetic oligonucleotides, then incubate at 65° C. for 6 hrs; (b) drysamples with speed-Vac; (c) dissolve the dried pellets in water; (d)desalt samples with Sephadex G-25 column; and (e) dry samples withspeed-Vac; (f) redissolve desalted samples in water.

Partially and completely deprotected oligonucleotides may becharacterized for further use or to verify procedures by any suitablemeans, including but not limited to gel electrophoresis, urea-acrylamidegel electrophoresis, 5′ end labeling with T4 polynucleoide kinase, HPLCanalysis, mass spectrometry, etc.

Suitable animals can be immunized with the oligonucleotides describedabove by parenteral injection of the oligonucleotide in a suitablecarrier, such as sterile saline solution. Injection may be by anysuitable route, including but not limited to subcutaneous,intraperitoneal, intravenous, intraarterial, intramuscular, etc.Suitable animals are typically mammals, including mice, rabbits, rats,etc.

In a particular embodiment, for the production of monoclonal antibodies,young female BALB/c mice are used, and the time course of injection ofthe antigen material is:

first day initial injection

14th day first boosting

28th day second boosting

4 day before fusion final boosting

Additional injections may be employed if desired. The antigen amount maybe 50 μg or 100 μg of oligonucleotides unprotected (for controlantibody) or protected, for each mouse per time. When, as preferred,beads or other solid support used as the support for oligonucleotidesynthesis are injected into the animal, the beads or particles aresuspended in water, then injected into mice. If a nucleotide solution isused, then the solution is mixed with complete or incomplete Freund'sadjuvant and injected into mice.

Polyclonal antibodies can be harvested from animals immunized orinnoculated as described above in accordance with known techniques, orspleen cells harvested from the animals, hybridoma cell lines producedfrom the spleen cells, and the hybridoma cell lines screened for theproduction of desired antibodies, also in accordance with knowntechniques.

Oligonucleotides that contain or do not contain biotin molecules at 3′or 5′ ends (for ELISA assay as described below) may be synthesized inaccordance with standard techniques. Examples are:

Poly Ibu-dG 20 mers (with or without biotin);

Poly Ibu-dA 20 mers (with or without biotin);

Poly Ibu-dC 20 mers (with or without biotin);

Poly Ipr-Pac-dG 20 mers (with or without biotin);

Poly Bz-dC 20 mers (with or without biotin);

Poly Bz-dA 20 mers (with or without biotin);

Poly dT 20 mers (with or without biotin);

Poly Pac-dA 20 mers (with or without biotin);

Poly Ac-dC 20 mers (with or without biotin); and

Poly dmf-G 20 mers (with or without biotin).

Antibodies produced as described above may be characterized by anysuitable technique to determine the binding properties thereof,including but not limited to Western blot and immunodot-blot.

In addition to the use of polyclonal and monoclonal antibodies, thepresent invention contemplates the production of antibodies byrecombinant DNA, or “antibody engineering” techniques. For example, mRNAisolated from hybridoma cells may be used to construct a cDNA libraryand the sequence encoding whole antibody or antibody fragments (e.g.,scFab or Fab fragments) isolated and inserted into suitable expressionvector, and the expression vector inserted into a host cell in which theisolated cDNA encoding the antibody is expressed.

Monoclonal Fab fragments may be produced in Escherichia coli byrecombinant techniques known to those skilled in the art. See, e.g., W.Huse, Science 246, 1275-81 (1989).

8. Screening of Antibodies

Screening sera and hybridoma cell culture media for protecting groupspecific antibodies may be carried out as follows:

A. Sera

1. Pre-immune (prior to immunization) sera are collected by standardmeans from the mice to be inoculated with protecting group conjugated toa solid support (directly or through an oligomer).

2. Post-innoculation sera are also collected.

3. An ELISA assay is performed in which the specific protecting groupremains on a biotinylated oligonucleotide conjugated to the microtiterplate. Other microtiter plate wells contain control oligomers that haveno protecting groups, or oligonucleotides with other protecting groups.The secondary antibody is a goat anti-mouse IgG with a conjugatedphosphotase for visualization of antibody

4. Those mice that have positive activity against the specificprotecting group are boosted and sacrificed for the production ofhybridomas.

B. Hybridoma Cell Culture Media

1. Approximately 1000 cultures are generated from each spleen hybridcell production.

2. Cultures are grown in microtiter plate wells, 96 well plates.

3. Culture medium is removed from each well and used in ELISA assays asdescribed above in which each of the ˜1000 microtiter plate wellscontain the protected oligonucleotide conjugated to the plate.

4. Those cultures producing antibody that has positive activity aretransferred to larger culture wells, 24 well microtiter plates.

5. Culture media from the larger cultures are re-tested for activityagainst the protecting group and are also assayed for specificity; ie.controls of no protecting group and of other protecting groups.

6. Those cultures that are positive are cloned out (diluted), re-testedand cloned out again to the point that each final culture must be theresult of one cell; ie. mono-culture. Media from these final culturesare thoroughly assessed for specificity and affinity. Specificity andaffinity are assessed using a dot-blot assay.

C. Dot-Blot Assays in Lieu of ELISA Assays

1. Antibodies against some protecting groups are not tractable to beingtested in the microtiter plate well environment and must be tested usinga dot-blot assay. One example is the 5′-terminal protecting group,dimethyl-trityl (DMT).

2. The Dot-blot assay on a nitrocellulose membrane is accomplished asdescribed elsewhere in the application for most purposes. However, thisis not possible in assessing antibody production by ˜1000 microtiterwell cultures with little media available. Thus, a novel adaptation hasbeen developed.

a) The protected oligonucleotide is attached in dots to thenitrocellulose using UV-crosslinking. With DMT, the presence of the5′-DMT on the membrane is confirmed by treatment of a dot with mildacid—the dot turns yellow-orange. The presence of the 3′-biotin can beconfirmed with a commercial avidin stain.

b) The membrane is blocked (see dot-blot assay).

b) The dry membrane dots are carefully marked (pencil) and “punched” outof the membrane.

c) Individual dots are added to the cell culture media in individualmicortiter plate wells and incubated.

d) The individual dots are removed and passed on through the washing,secondary antibody, phosphotase reaction and color development usingmicrotiter plate wells with the appropriate reagents.

e) Those dots that are positive are related back to the originalmicrotiter plate well cultures from which the small amount of culturemedia was obtained.

f) Further culturing and cloning is accomplished as described in B.

9. Testing of Microarrays

The present invention may be used to test or screen oligonucleotidesthat are immobilized on a solid support such as a microarray forinsufficient deprotection or elongation of the oligonucleotidessynthesized thereon.

Solid supports used to carry out the present invention are typicallydiscrete solid supports. Discrete solid supports may be physicallyseparate from one another, or may be discrete regions on a surfaceportion of a unitary substrate. Such “chip-type” or “pin-type” solidsupports are known. See, e.g., U.S. Pat. No. 5,143,854 to Pirrung; U.S.Pat. No. 5,288,514 to Ellman (pin-based support); U.S. Pat. No.5,510,270 to Fodor et al. (chip-based support). Additional non-limitingexamples of oligonucleotide arrays which may be used to carry out thepresent invention, and methods of making the same, include but are notlimited to those described in U.S. Pat. Nos. 5,631,734; 5,599,695;5,593,839; 5,578,832; 5,510,270; 5,571,639; 6,056,926; 5,445,934; and5,703,223. Such devices may be used as described therein to carry outthe instant invention.

The solid support or substrate from which the array is formed may becomprised of any suitable material, including silicon. Theoligonucleotides may be polymerized or grown in situ from monomers (orindividual nucleotides) in situ on the microarray (in which case none ofthe currently available techniques for detecting protecting groups wouldbe useful for detecting incomplete deprotection or elongation of theoligonucleotides on the array, as one cannot pass the solid supportthrough an analytical device) or the oligonucleotides may be polymerizedseparately and then linked to the appropriate regions of the solidsupport. The array may include any number of different oligonucleotidesin different separate and discrete regions thereon, examples includingarrays of at least 1,000, at least 2,000, at least 10,000, or at least20,000 different oligonucleotides in different separate and discreteregions.

In general, a method of screening an oligonucleotide array forinsufficient deprotection or insufficient elongation of oligonucleotidestherein comprises the steps of:

(a) providing an oligonucleotide array as described above;

(b) providing an antibody as described above (that is, an antibody thatspecifically binds to a synthetic oligonucleotide having an organicprotecting group covalently bound thereto, which antibody does not bindto said synthetic oligonucleotide when said organic protecting group isnot covalently bound thereto). Preferably the antibody is one thatspecifically binds to an oligonucleotide having a protecting group,where the protecting group was employed in the course of the organicsynthesis of oligonucleotides carried by that array. Then;

(c) contacting said oligonucleotide array to said antibody to therebydetect the presence of insufficient deprotection or insufficientelongation of oligonucleotides therein. Such detection, which may bequalitative or quantitative, may be carried out by any suitableimmunoassay technique as described above.

In the method, steps (b) to (c) may be repeated at least once, with adifferent antibody on each repetition, so that a plurality of differentprotecting groups which may be present on oligonucleotides in the arraymay be detected.

Preferably, once insufficient deprotection (the presence of protectinggroups) in oligonucleotides in one or more (e.g., plurality) of theseparate and discrete regions is detected, the method further comprisesgenerating a record or indicia recording the presence of insufficientdeprotection or insufficient elongation of oligonucleotides in the leastone separate and discrete location (or plurality of separate anddiscrete locations) on the array. The indicia may be a qualitative orquantitative indicia of insufficient deprotection (includinginsufficient elongation).

The foregoing methods provide a correctable oligonucleotide array asillustrated in FIG. 11. The array comprises, in combination:

(a) a substrate 30 having a plurality of different oligonucleotidesimmobilized thereon, with the different oligonucleotides immobilized indifferent separate and discrete locations 31 on said substrate; and

(b) a plurality of indicia associated with said array, these indiciarecording the presence of insufficient deprotection or insufficientelongation of a plurality of different oligonucleotides, said differentoligonucleotides located in separate and discrete locations on saidarray. These indicia may be printed in a region of the array 32 by atechnique such a microlithography, printed on conventional medium suchas paper and shipped with the array, stored in a memory or memory deviceconnected to or formed on the array chip (which may be incorporated atlocation 32), provided in a separate data or computer file which may beprovided on a computer-readable medium such as a floppy diskette orCD-ROM, stored on a web site on the world wide web for downloading bythe end user of the array, etc. When the indicia are provided in aseparate data file, the array preferably further includes an identifiersuch as a code number formed on, connected to or associated with thearray (e.g., printed on a package containing the array, or on aninformation sheet packaged with the array, and/or printed directly onthe array). The indentifer may then be associated with the separateindicia (e.g., printed on a data sheet, used as a pass-word, fileidentifier and/or access code for the computer file, etc.) to insure thecorrect indicia containing the record of insufficient deprotectionand/or elongation are ultimately associated with the array by theultimate end user of the array.

A data device or memory device connected to the array may be carried outin accordance with known techniques, as described in U.S. Pat. Nos.5,925,562; 6,017,496; 5,751,629; and 5,741,462, and such devices used asdescribed therein to carry out the instant invention.

The end user of the array may utilize the indicia described above tocompensate for insufficient deprotection or insufficient elongation ofoligonucleotides on said array in a method comprising:

(a) providing a substrate as described above.

(b) providing at least one, or a plurality of, indicia associated withsaid array as described above.

(c) providing a test compound. The test compound may be a member of alibrary of test compounds, and may be any suitable compound such as aprotein; peptide or oligonucleotide (e.g., a DNA or RNA, such as mRNA);and then

(d) detecting the binding of said test compound to at least one of saidplurality of different oligonucleotides (e.g., by contacting the testcompound to the array); and then

(d) detecting determining the degree of binding (including simply thepresence or absence of binding) of the test compound to one or moreoligonucleotides on the array from (i) said detected binding and (ii)said indicia recording the presence of insufficient deprotection orinsufficient elongation. Thus, insufficient deprotection or insufficientelongation of oligonucleotides in one or more locations in the array maybe compensated for during the determining step. Such compensation may beachieved by any means, including ignoring particular separate anddiscrete regions on the array (e.g., in favor of other separate anddiscrete regions of the array that contain the same oligonucleotide). Inanother example, if one or more locations contain insufficientdeprotection or elongation such that binding to those locations isreduced, the binding data derived from an experiment with that array canbe adjusted upwards for those locations to indicate greater binding thanthat which would otherwise be indicated without the control madepossible by the recorded indicia. The detecting or determining step maybe carried out by any suitable means, such as generating a colorindication of degree of binding, generating a numeric indication ofdegree of binding, generating a graphic or other symbolic indication ofdegree of binding, etc. The degree of binding may be an indication ofbinding is binding affinity, binding amount, or both binding affinityand binding amount, but is typically an indication of the amount of testcompound that binds to a particular separate and discrete region of thearray.

The present invention is explained in greater detail in the followingnon-limiting Examples.

Example 1 Synthesis of Oligonucleotides

Synthesis was performed on an ABI DNA/RNA Synthesizer, Model 394 (PEBiosystems, 850 Lincoln Centre Drive, Foster City, Calif. 94404)according to manufactories protocol. Slightly modified 1 micromolarscale cycle was used during synthesis (see manufacturer's instructions).The primary starting materials (and suppliers/manufacturers inparentheses) were as follows:

Activator (0.45 M tetrazole in acetonitrile), CAP A (aceticanhydride/tetrahydrofuran/2,6 lutidine), CAP B (N-methylimidazole/tetrahydrofuran) and oxidizer (0.02 M iodine/pyridine/THF/H2O)(Prime Synthesis)

Pac-dA (5′-dimethoxytrityl-N-phenoxyacetyl-2′-deoxyAdenosine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research)

Ipr-Pac-dG (5′-dimethoxytrityl-N-p-isopropyl-phenoxyacetyl-2′-Guanosine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research)

Ac-dC (5′-dimethoxytrityl-acettyl-2′-deoxycytidine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research)

dmf-G (5′-dimethoxytrityl-dimethylformamidine-Guanosine,2′-O-TBDMS-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (GlenResearch)

Bz-dC---CPG beads (5′-dimethoxytrityl-N-benzoyl-2′-deoxycytidine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite-succinyllinker-beads (3000 Ang) (CPG Inc.)

Ibu-dG---CPG beads (5′-dimethoxytrityl-N-isobutyl-2′-deoxycytidine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite-succinyllinker-beads (3000 Ang) (CPG Inc.)

The following compounds were synthesized, the compounds being linked tobeads as shown:

-   -   Pac-dA---Pac-dA---Bz-dC---succinyl linker---Beads    -   Pac-dA---Pac-dA---Ibu-dG---succinyl linker---Beads    -   Ipr-Pac-dG---Ipr-Pac-dG---Bz-dC---succinyl linker---Beads    -   Ipr-Pac-dG---Ipr-Pac-dG---Ibu-dG---succinyl linker---Beads    -   Ac-dC---Ac-dC---Bz-dC---succinyl linker---Beads    -   Ac-dC---Ac-dC---Ibu-dG---succinyl linker---Beads    -   dmf-G---dmf-G---Bz-dC---succinyl linker---Beads    -   dmf-G---dmf-G---Ibu-dG---succinyl linker---Beads

The foregoing compounds were administered directly to animals as animmunogen, without separating the oligonucleotide from the solidsupport, for the production of antibodies, as further described inExample 2 below.

Example 2 Innoculation of Animals

Female BALB/c mice of eight to twelve weeks old were purchased fromCharles River, Raleigh, N.C., USA. The mice were housed in cases withfilter caps.

After oligonucleotide chain synthesis was completed as described inExample 1, the beads with nucleotides were gently crushed byhand-pressuring the glass plates, between which beads were positioned.

5 μM of each eight oligonucleotides mentioned above were mixed in 4 mlPBS (150 mM sodium chloride in 100 mM phosphate buffer, pH 7.2).

The mixture was thoroughly vortexed suspending the crushed beads. 150 μLof the vortexed mixture was taken and added into 300 μL of PBS in asyringe. Just before injection, the solution containing beads was mixedagain by shaking the syringe to suspend the broken beads. Then 150 μL or300 μL of well-mixed solution was injected into mouse peritoneal cavity.This procedure was used for the first injection and the followingboosts.

Injection Time Schedule:

Injection Date (day) first  0 second  14th third  28th  4th  42nd  5th 56th  6th  70th  7th  84th  8th  98th  9th 112th 10th 138th 11th(final, 4 day before fusion) 142nd

Four days after the final injection, spleen cells are harvested from theanimals and fused with myeloma cells (P3x.63.Ag8.653) in accordance withknown techniques to produce hybridoma cell lines, which are thenscreened to determine the binding characteristics as described below toisolate particular cell lines that produce the desired antibody of theinvention.

Example 3 Immunodot-Blot Assay for Antibody Characterization

The Immunodot-blot assay involves UV cross linking of oligonucleotidesonto membrane paper, and is directly applicable to a test kit fordetection, identification and quantifying the protecting groups onproduct oligomers. This procedure may be carried out as follows: (a) wetmembrane paper with TBS (10 mM Tris, pH 7.2; 150 mM NaCl); (b) blotoligonucleotides to be tested onto membrane paper under vacuum; (c) UVcross link nucleotide onto membrane paper; (d) block membrane paper with1% casein-TBST (TBS plus Tween 20, 0.1% by volume) at room temperaturefor 2 hr or 4° C. overnight; (e) wash membrane with TBST 3 times, eachfor 15 min; (f) form antigen-antibody complex by incubation of platewith sample be tested (diluted in 1% casein-TBST) at room temperaturefor 1 hr; (g) wash as above; (h) react with second antibody conjugate(diluted in 1% casein-TBST) at room temperature for 1 hr; (i) wash asabove; 6) develop color reaction by incubation of membrane withsubstrate solution.

Example 4 Dot-Blot Assay of Monoclonal Antibody 1 H11

Monoclonal antibody 1 H11, produced as described in Example 2 above, wascharacterized by a dot-blot assay as described in Example 3 above.Results are shown as a bar graph in FIG. 1. In FIG. 1, lanes (orcolumns) 1 and 2 represent oligoPac-dA20mers treated with NH₄OH for 6hours at 65° C. and 15 minutes at 4° C., respectively. Columns 3 and 4represent oligoBz-dC20mers treated with NH₄OH for 6 hours at 65° C. and15 minutes, respectively. Columns 5 and 6 represent oligoAc-dC20merstreated with NH₄OH for 6 hours at 65° C. and 15 minutes, respectively.Columns 7 and 8 represent oligoIpr-Pac-dG20mers treated with NH₄OH for 6hours at 65° C. and 15 minutes, respectively. Columns 9 and 10 representoligoIbu-dG20mers treated with NH₄OH for 6 hours at 65° C. and 15minutes, respectively. Columns 11, 12 and 13 represent oligodT20mers,completely deprotected, with DMT group only, and with cyanoethyl grouponly, respectively. Antibody activity is given as optical density (479nm) from ELISA (Example 7 below), and the positive or negative result ofthe dot blot assay is given in the open or filled circle appearing overeach column in the bar graph. Note the activity of monoclonal antibody 1H11 in selectively binding to the oligoIbu-dG20mer in column 10.

Example 5 Dot-Blot Assay of Monoclonal Antibody 7 H3

Monoclonal antibody 7 H3, produced as described in Example 2 above, wascharacterized by a dot-blot assay as described in Example 3 above.Results are shown as a bar graph in FIG. 1. In FIG. 1, lanes (orcolumns) 1 and 2 represent oligoPac-dA20mers treated with NH₄OH for 6hours at 65° C. and 15 minutes at 4° C., respectively. Columns 3 and 4represent oligoBz-dC20mers treated with NH₄OH for 6 hours at 65° C. and15 minutes, respectively. Columns 5 and 6 represent oligoAc-dC20merstreated with NH₄OH for 6 hours at 65° C. and 15 minutes, respectively.Columns 7 and 8 represent oligoIpr-Pac-dG20mers treated with NH₄OH for 6hours at 65° C. and 15 minutes, respectively. Columns 9 and 10 representoligoIbu-dC20mers treated with NH₄OH for 6 hours at 65° C. and 15minutes, respectively. Columns 11, 12 and 13 represent oligodT20mers,completely deprotected, with DMT group only, and with cyanoethyl grouponly, respectively. Antibody activity is given as optical density asdescribed above, and the positive or negative result of the dot blotassay is given in the open or filled circle appearing over each columnin the bar graph. Note the activity of monoclonal antibody 1 H11 inselectively binding to the oligoBz-dC20mer in column 4.

Example 6 Western Blot Assay for Antibody Characterization

The Western blot assay involves low voltage transfer of oligonucleotidesfrom gel to membrane paper and UV cross linking of oligonucleotides ontothe membrane. This assay may be carried out as follows: (a) cast 15%non-denaturing gel containing 10 mM MgCl; (b) load oligonucleotides(oligomers) into the wells of the gel; (c) run gel at 200 voltage in icebath; (d) transfer oligonucleotides from gel to membrane paper at 25voltage for 25 min in ice bath; (e) UV cross link polynucleotides onmembrane; (f) block membrane paper with 1% casein-TBST at roomtemperature for 2 hr or 4° C. overnight; (g) wash membrane with TBST 3times, each for 15 min; (h) incubate samples be tested (diluted in 1%casein-TBST) at room temperature for 1 hr; (i) wash as above; (j)incubate membrane with second antibody conjugate (diluted in 1%casein-TBST) at room temperature for 1 hr; (k) wash as above; and (l)color-develop by incubation of membrane with substrate solution.

Example 7 Detection of Antibody Using Biotinylated Polynucleotides asAntigen and an ELISA Involving Streptavidin-Biotin System

An enzyme-linked immunosorbent assay (ELISA) for the detection of theantibody is carried out as follows: (a) pre-screen microtiter plate thatis pre-coated with streptavidin; (b) coat the plate with a preparationof biotinylated oligonucleotide or other materials to be tested (at 5μg/ml in PBS) (PBS: 150 mM NaCl, 10 mM Phosphate buffer, pH 7.4), thenincubate at room temperature for 2 hrs; (c) wash 3 times with 0.1% Tweenin PBS (PBST), each for 15 min; (d) block with 1% casein in PBST at roomtemperature for hrs or 4° C. overnight; (e) wash as above; (f) formantigen-antibody complex by incubation of plate with antibody (orantibodies) at room temperature for 1 hr; (g) wash as above; (h) reactwith second antibody-peroxidase conjugate (in 1% casein-PBST) at roomtemperature for 1 hr; (i) wash as above; (j) develop color reaction byadding tetramethylbenzidine (TMB) solution (TMB solution: 42 mM TMB,0.004% H₂O₂, 0.1 M acetate buffer, pH 5.6) and incubating at roomtemperature for 15 min, then stop the reaction with 2 M H₂SO₄; and (k)read absorption value at 469 nm.

Example 8 ELISA and Dot-Blot Assay of Monoclonal Antibody AgainstBenzoyl, Isobutryl, and Isopropylphenoxyacethyl

Monoclonal antibodies (mAb) against protecting groups benzoyl (Bz),isobutryl (ibu), and isopropylphenoxyacethyl (ipr-Pac), produced asdescribed in Example 2 above, were characterized by a standard ELISAassay and a dot-blot assay as described in Example 3 above. An ELISAassay developed with biotinylated nucleic acids of 20 residues eachattached to a 96-well microtiter plate demonstrated the specificity ofthe antibodies for their respective antigens. FIG. 3A, FIG. 4A, and FIG.5A show results for monoclonal antibodies against Bz, ibu, and ipr-Pac,respectively. The figures show completely deprotected (<1% Bz remaining)homopolymers of dC residues, designated oligo dC(Bz), ie. originallyprotected with Bz (lane 1, open bar), protected (>97% Bz remaining)oligo Bz-dC (lane 2, shaded bar), completely (<1% ipr-Pac remaining)deprotected oligo dG(ipr-Pac) (lane 3), protected (>76% ipr-Pac) oligoipr-PacdG (lane 4), completely (<1% ibu remaining) deprotected oligodG(ibu) (lane 5), protected (>91% ibu remaining) oligo ibu-dG (lane 6),and completely deprotected oligo dT (lane 7). The dT polymer had but oneprotecting group, dimethyltrityl (DMT) that was removed from the 5′OH ofthe 5′-terminal residue with mild acid. Finally, lane 8 of shows oligodT with DMT remaining.

Dot-Blot assays of anti-Bz mAb, anti-ibu mAb, and anti-ipr-Pac mAbactivities were performed in which the 20mer DNAs were linked tonitrocellulose membrane by UV. The amounts of 20mer DNA applied to themembrane are shown to the right of FIG. 3B, FIG. 4B, and FIG. 5B anddemonstrate the level of sensitivity of the assay. The DNAs used to testanti-Bz mAb were those described for the ELISA plus deprotected oligodA(Bz), protected oligo Bz-dA, oligo dC(ibu), oligo ibu-dC, oligodA(ibu) and oligo ibu-dA. FIG. 3B shows that the anti-Bz mAb recognizedthe protecting group on dA and dC. The DNAs used to test anti-ibu mABwere those described for the ELISA plus protected oligo ibu-dA,deprotected oligo dA(ibu), oligo ibu-dC, oligo dC(ibu) and all are notedat the top of the dot-blot. FIG. 4B shows that the anti-ibu mAbrecognized ibu on dG, the most common use of the protecting group, butalso on dA. The DNAs used to test anti-ipr-Pac mAb were those describedfor the ELISA plus protected oligo ibu-dA, deprotected oligo dA(ibu),oligo ibu-dC, oligo dC(ibu), oligo Bz-dA, oligo dA(Bz) and all are notedat the top of the dot-blot. FIG. 5B shows that the anti-ipr-Pac mAbrecognized ipr-Pac on dG, the most common use of the protecting group,but also on dA and dC. The mAb also recognized the ibu protecting group(ibu-dG, ibu-dA and ibu-dC). This cross-reactivity indicates that theantibody was highly selective in its identification of a chemistrycommon to both ipr-Pac and ibu, possibly CH(CH₃)₂. Thus the anti-ibu andanti-iprPac mAbs could be used in combination to identify the protectinggroup remaining on an oligo.

Greater amounts of DNA were tested in a dot blot assay of anti-ibu mAb(FIG. 4C). The results of this experiment demonstrated that the ibuprotecting group was recognized by the mAb no matter which nucleobasewas protected.

FIG. 3C, FIG. 4D, and FIG. 5C demonstrate that partially deprotectedoligomers can be re-treated to remove the remaining protecting groups,and re-tested with mAb. FIG. 3C shows that anti-Bz mAb recognizedre-deprotected oligomer oligo Bz-dC (center column). Likewise, FIG. 4Dshows that anti-ibu mAb recognized re-deprotected oligomer oligo ibu-dG(center column) and FIG. 5C shows that anti-ipr-Pac mAb recognizedre-deprotected oligomers oligo ipr-Pac-dG and oligo ibu-dG (columnssecond from left and forth from left, respectively). Thus, this approachis applicable to quality control without having to discard expensivenucleic acid samples.

An RNA standard with protecting groups Bz, ibu and ipr-Pac wassynthesized and assayed for identification of the protecting groups withthe mAb against Bz (FIG. 3D), ibu (FIG. 4E), and ipr-Pac (FIG. 5D).Dot-blot assays clearly show that the monoclonal antibodies do notdifferentiate RNA from DNA. Although there was a higher backgroundsignal with RNA than with DNA, there was a significant distinctionbetween RNA with and without protecting groups, especially at the loweramounts of RNA. The amount of RNA on the membrane was estimated from theoptical absorbance of the sample.

Example 9 mAb Dot-Blot Assay of Protecting Groups vs HPLC

Dot-blot detection of Bz groups remaining on a standardized 20mer oligodC molecule were performed as described in Example 3. Completelydeprotected and the untreated oligo dC 20mers were analyzed for the Bzprotecting group using a totally independent and differentquantification method. The two oligomers were hydrolyzed to theconstituent nucleosides and then their nucleoside composition identifiedand quantified using a recognized high performance liquid chromatography(HPLC) method with concentrated samples. Because of the lack ofsensitivity, HPLC detection required 50-100 fold the amounts of Bz-dCused in the mAb assays (see FIG. 7). FIG. 6A shows the result of anti-BzmAb tested against nmole amounts of Bz groups on protected oligo Bz-dC(right column) and the same nmole amounts of Bz- on Bz-dC (left column).Each amount of Bz-dC oligo was diluted with completely deprotected dColigo of the same length (20mer) to demonstrate the sensitivity of themAb detection even in the presence of 2500-fold dC (ie. 0.04%). The mAbassay demonstrated that the mAb could detect the Bz group on DNA even inthe presence of a 2500-fold excess of dC in DNA.

The dot-blot shown in FIG. 6A was subjected to densitometry toquantitate the mAb response. After background subtraction, the remainingdensity was plotted as a function of Bz groups in oligo Bz-dC determenedby HPLC (FIG. 6B). The data indicated that the high sensitivity of theanti Bz mAb detection was linear in 0.1-1.0 nmol range.

Next, it was determined whether the mAb response could be enhanced withan increase in the amount of DNA on the dot-blot membrane. The amount ofBz was determined by standard HPLC methods. This experiment showed thatdetection of the Bz protecting group in a mixture of the protectedsample with the deprotected sample at a ratio of 1/2500 could beenhanced by increasing the amount of DNA on the membrane, though theratio was maintained (FIG. 6C).

Finally, experiments were conducted to show a direct comparison of themAb and HPLC detection of Bz. Anti-Bz mAb was utilized in a dot-blotassay to detect Bz on dC in the oligo Bz-dC (20mer). The densityresponse of the Bz group detected Bz by the mAb assay and quantified bydensitometry was plotted against the amount of Bz in the DNA on each dot(FIG. 7A). The amount of Bz in the DNA was calibrated by digestion of alarge amount of DNA and analysis by HPLC identification andquantification of the Bz-dC mononucleoside. For HPLC experiments, threesamples of Bz-dC oligo were hydrolyzed and analyzed for composition byHPLC. The response of the UV-diode array detector was plotted againstthe amount of Bz in the samples (FIG. 7B). The sample amounts weredetermined by comparison to samples “spiked” with known amounts ofBz-dC. The amounts of Bz-dC added to samples as spikes were from aweighed stock of Bz-dC. Thus, the HPLC response was calibrated withknown amounts of Bz-dC. The results of these experiments show that thedetection of Bz by anti-Bz mAb was within the pmole range whereas HPLCdetection of Bz was limited to the nmole range.

Example 10 Detection of Remaining Protecting Groups in CommercialSamples

A blind study was conducted to demonstrate the detection of remainingprotecting groups in commercial samples by mAb. The purpose of the thisexperiment was to determine if protecting groups could be detected andidentified with mAb technology in presumably completely deprotectedsamples that had been treated as commonly accomplished in the oligosynthesis industry. The nature of the protecting groups used by eightselected companies was not known, thus the experiment was a blind study.Two 20mer oligos (oligo dA-dC and oligo dG-dT) from each of the eightcompanies were ordered to be synthesized and deprotected, and saltremoved under as identical conditions as possible. The oligos wereshipped by express mail, as is often the case, and then subjected to mAbanalysis by dot blot. The dA-dC oligo from one company (#6), andpossibly a second (#2), had remaining Bz protecting groups as determinedby anti-Bz mAb testing (FIG. 8A). The dG-dT oligos from two companies(#2 and #6) had ipr-Pac protecting groups remaining as determined byanti-ipr-Pac mAb (FIG. 8B). The remaining protecting groups in thecommercial samples were confirmed by increasing amounts of sample andfurther deprotection and re-analyses. The oligo dA-dC samples fromcompanies #2 and #6 were tested in higher amounts to confirm thepresence of the Bz protecting group. In addition, the samples weretreated to remove the remaining protecting groups using a standardprotocol. The re-analysis after further deprotection indicated that thegroups were now removed (FIG. 8C). This also demonstrates that expensivenucleic acid samples can be re-treated to remove protecting groups andthat they need not be discarded. The oligo dG-dT samples were re-treatedto remove remaining protecting groups and re-analyzed with anti-ipr-PacmAb with the result that the ipr-Pac group could be removed withoutsacrificing the DNA (FIG. 8D).

Example 11 Polyclonal Antibody Against Dimethyltrityl

Production and analyses of polycolonal antibody against the 5′ terminalprotecting group, dimethyltrityl (DMT) were as described in Example 2.Four mice were inoculated with DMT and sera were drawn from the miceafter some weeks of boosting with antigen. DMT [DMT-OH], three DMT atthe 5′-end of the deoxynucleotide trimer d(T)₃[(DMT)₃-d(T)₃], three DMTat the 5′-end of the deoxynucleotide 20mer d(T)₃ with 3′-biotin[(DMT)₃-d(T)₂₀-biotin], one DMT at the 5′-end of the deoxynucleotide20mer d(T)₂₀ with 3′-biotin [DMT-d(T)₂₀-biotin], the dT 20mer with3′-biotin [d(T)₂₀-biotin], one DMT with biotin [DMT-biotin] andtris-borate saline control were applied to a nitrocellulose membranethat was then assayed with mouse sera (inoculated mice #1-4 and acontrol serum, normal) to assess anti-DMT antibody, mild acid to revealpresence of the DMT (TBS), and avidin to reveal the presence of biotin(FIG. 9). Sera from mice #2 and #4 recognized DMT [as (DMT)₃-d(T)₃],whereas mice #1, #3, and the normal mouse did not. Mild acid revealedthe presence of DMT as a yellow color (not visible in figure) and avidinrevealed the presence of biotin.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1-25. (canceled)
 26. A method of making an antibody that specificallybinds to a synthetic oligonucleotide having a organic protecting groupcovalently bound thereto, which antibody does not bind to the saidsynthetic oligonucleotide when said organic protecting group is notcovalently bound thereto, said method comprising the steps of:synthesizing said synthetic oligonucleotide on a solid particulatesupport with said organic protecting group covalently bound to saidsynthetic oligonucleotide, or synthesizing a nucleotide on said solidsupport with said organic protecting group bound to said nucleotide; andthen, without removing said oligonucleotide or nucleotide from saidsolid support; immunizing an animal with said synthetic oligonucleotideor nucleotide bound to said solid support in an amount sufficient toproduce said antibody.
 27. A method according to claim 26, wherein saidsynthesizing step is followed by the step of fragmenting said beadsprior to said immunizing step.
 28. A method according to claim 26,further comprising the step of: collecting said antibody from saidanimal.
 29. A method according to claim 26, further comprising the stepsof: collecting spleen cells from said animal; then producing a pluralityof hybridoma cell lines from said spleen cells; and then isolating aparticular hybridoma cell line that produces said antibody from saidplurality of hybridoma cell lines.
 30. A method according to claim 26,wherein said synthetic oligonucleotide is covalently bound to said solidsupport.
 31. A method according to claim 26, wherein said syntheticoligonucleotide is covalently bound to said solid support with asuccinyl linker.
 32. A method according to claim 26, wherein said solidsupport comprises a controlled pore glass bead. 33-55. (canceled)